SV40 large T antigen (LT) is a potent carcinogen, and plays an essential role for SV40 DNA replication as the replicative helicase and replication initiator protein. SV40 replication serves as a model for eukaryotic DNA replication, as SV40 uses all the essential cellular replication proteins (primase, polymerase, PCNA, Topoisomerases, etc.), except for the helicase and cellular initiator proteins that consist of multiple initiator factors (such as Orc, Ctd1, Cdc6, MCM in eukaryotes, or DnaA/DnaC/DnaB in prokaryotes) to initiate DNA replication, i.e. marking the replication origin, recruiting helicase, melting origin, and activating helicase. For SV40 replication, LT alone fulfills essentially all the initiator functions and is the helicase for replication fork unwinding during elongation phase. The long-term goal of this research is to understand how LT functions as a helicase to coordinate the functions of the other replication proteins for DNA replication, as well as how LT transforms cells. Specific aims are designed to understand how LT hexameric and double hexameric helicase melt the origin DNA and unwinds dsDNA to initiate DNA replication. We plan to use mainly X-ray protein crystallography, assisted with EM and AFM, single molecule assay, computational method, molecular biology and functional biochemistry in vitro and in cells. The results from this research are expected to have potential impact on the field of DNA replication in eukaryotic cells and on cancer biology.